Discovery of chromosomal factors contributing to the virulence of Yersinia pseudotuberculosis, including Fis, a regulator of resistance to reactive oxygen species.

Abstract: All three pathogenic Yersinia species share a conserved virulence plasmid that encodes a Type 3 Secretion System (T3SS) and its effectors. This system contributes to growth and virulence in mammalian infection by injecting effector proteins into innate immune cells to dismantle their bactericidal functions. However, in the absence of this plasmid, the enteric pathogen Yersinia pseudotube... read morerculosis (Yptb) retains the ability to colonize and grow within host organs. To uncover chromosomal factors that contribute to pathogenicity of Yptb in the absence of the virulence plasmid, we utilized a high-throughput, transposon screen (TnSeq). More than 30 genes, including many that were previously uncharacterized, were identified. Next, I developed a deep sequencing-based approach to validate and further characterize the phenotype of 18 of those genes in both WT and plasmid-deficient Yptb strains by infecting mixed pools of in-frame knockouts into immunocompetent mice. Fourteen mutants in the plasmid-deficient strain and 13 in the WT strain were attenuated for growth in livers, indicating that most of these factors were essential, even in the presence of the virulence plasmid. The growth of these mutants was assessed in immunocompromised mice to determine which of these factors disarm host defenses produced by innate immune cells. Mutants containing deletions of the dusB-fis operon, which encodes the nucleoid-associated protein Fis, were restored for growth in mice lacking neutrophils and inflammatory monocytes, two of the major cell types responsible for controlling Yersinia infection. This operon was not important for secretion or translocation of T3SS effectors, but protected Yptb from oxidative stress by regulating the transcription of several ROS-detoxifying genes. Strikingly, this protection was critical for virulence, as mice unable to produce ROS were more sensitive to infection by dusB-fis than WT mice. Notably, this is the first report of the requirement for Fis during Yersinia infection and also highlights a novel mechanism by which Yptb defends against ROS in mammalian tissues. read less